Hormones of Energy Metabolism in Critically Ill Foals: Insulin, Glucagon,
Leptin, Adiponectin, Ghrelin and Growth Hormone
THESIS
Presented in Partial Fulfillment of the Requirements for the Degree Master of Science in
the Graduate School of The Ohio State University
By
Rosa Jessica Irene Maria Barsnick
Graduate Program in Veterinary Clinical Sciences
The Ohio State University
2010
Master's Examination Committee:
Assistant Professor Ramiro Toribio, Advisor
Professor Catherine Kohn
Assistant Professor Margaret Mudge
Copyright by
Rosa Jessica Irene Maria Barsnick
2010
Abstract
Endocrine dysregulation of energy metabolism is well documented in critically ill
humans, but limited information exists in septic foals. The purpose of this study was to
provide information on energy metabolism hormonal response in critically ill foals,
focusing on insulin, glucagon, leptin and adiponectin, as well as ghrelin and growth
hormone (GH), and to determine the association of these hormones with survival in
septic, sick non-septic and healthy foals.
We hypothesized that concentrations of insulin, glucagon, leptin, ghrelin, GH and
triglycerides will be higher, while adiponectin and glucose will be lower in septic foals
than healthy and sick non-septic foals. Magnitude of these differences will be associated
with severity of disease and non-survival. This study was a prospective multi-center
cross-sectional study, and 44 septic, 62 sick non-septic, and 19 healthy foals of <7 days
of age were included. Blood samples were collected at admission. Foals with positive
blood culture or sepsis score ≥12 were considered septic.
Septic foals had lower glucose and insulin, and higher triglyceride and glucagon
concentrations than healthy foals. Glucagon and adiponectin concentrations were not
different between septic foals that died (n = 14) or survived (n = 30). Higher insulin and
lower leptin concentrations were associated with mortality. Insulin sensitivity assessed by
QUICKI (quantitative insulin sensitivity check index) was increased in septic foals.
ii
Septic foals had significantly higher ghrelin concentrations than sick non-septic foals and
healthy controls. GH was higher in hospitalized foals (septic and sick non-septic)
compared to healthy foals. Both hormones were negatively correlated with glucose and
positively correlated with triglycerides. There was no difference in ghrelin and GH
concentrations between septic foals that died (n = 14) or survived (n = 30), but higher
ghrelin concentrations were associated with higher sepsis scores.
The endocrine energy response to systemic inflammation and negative energy balance in
septic foals is characterized by hypoglycemia, hypertriglyceridemia, low insulin and high
glucagon concentrations. Energy endocrine response, especially leptin and insulin, differs
between septic foals and critically ill humans. Increases in ghrelin and GH also appear to
be associated with the energy status in these foals.
iii
Dedicated to my friends in Wisconsin who are like family to me.
iv
Acknowledgments
I want to thank Ramiro Toribio for his mentorship and guidance in all aspects of this
research project and what else was required to successfully complete a master’s program.
Special thanks go to Phoebe Smith who always stood in for me when research seemed to
overwhelm me. I am endlessly grateful for her mentorship in the clinical setting of my
residency.
I want to acknowledge Catherine Kohn’s support and advice and especially her
commitment to the graduate program; and I want to thank Margaret Mudge for her
expertise and for filling in for Phoebe Smith on the examination committee.
Thanks go out to all of the clinicians and technical staff at Hagyard Equine Medical
Center in Lexington, KY, as well as the Galbreath Equine Center for their support of this
project. I also found myself lucky to have Holly Brown, Sam Coe, Brandy Marlow and
Krista Hernon to help me in the lab and with data retrieval.
This research was funded by the Morris Animal Foundation – thank you for choosing our
project.
v
Vita
since
07/07
Residency and Graduate Teaching and Research Assistant,
Veterinary Clinical Sciences, The Ohio State University,
Columbus, OH, USA
10/06 - 06/07
Clinical instructor, Equine Clinic, Internal Medicine, JustusLiebig-University of Giessen, Germany
04/06 - 07/06
Internship, Wisconsin Equine Clinic and Hospital,
Oconomowoc, WI, USA
Associate, Veterinary practice for horses and small animals,
C. Schweer, Isingerode, Germany
Associate, Bayreuth Equine Clinic, Dr. W. Schill, Eckersdorf,
Germany
Doctorate (Dr. med vet.)
Department of Animal Nutrition, School of Veterinary Medicine
Hanover, Germany
02/06 - 09/06
02/04 - 12/05
12/01 - 12/03
11/00 - 08/02
10/94 - 06/00
Associate, Veterinary practice for horses and small animals,
Dr. S. Knorr, Goslar, Germany
School of Veterinary Medicine Hanover, Germany
vi
Publications
Barsnick R, Investigation on acceptability and digestibility of differently conditioned
dried sugar beet pulp in the horse, Doctoral Thesis 2003.
Barsnick R, Köhler K, Hunsiga M, Fey K. Pancreatic adenocarcinoma in a warmblood
gelding. Tierärztliche Praxis, Ausgabe G 2008; 36(4):273-277.
Fields of Study
Major Field: Veterinary Clinical Science
vii
Table of Contents
Abstract .......................................................................................................................... ii
Acknowledgments ...........................................................................................................v
Vita ............................................................................................................................... vi
List of Tables................................................................................................................. ix
List of Figures ............................................................................................................... xi
Chapter 1: Introduction and Literature Review ................................................................1
Chapter 2: Insulin, glucagon, leptin and adiponectin in critically ill foals .........................6
2.1
Materials and Methods ......................................................................................6
2.2
Results............................................................................................................. 11
2.3
Discussion ....................................................................................................... 15
Chapter 3: Ghrelin and Growth Hormone in Critically Ill Foals ..................................... 28
3.1
Material and Methods ...................................................................................... 28
3.2
Results............................................................................................................. 31
3.3
Discussion ....................................................................................................... 34
References .......................................................................................................................4
viii
List of Tables
Table 2.1. Serum glucose, triglyceride, hormone concentrations and QUICKI in neonatal
foals at admission (n = 125) ........................................................................................... 21
Table 2.2. Correlation (rs) between blood glucose and triglyceride concentrations,
QUICKI and hormones (healthy foals only, n = 19) ....................................................... 21
Table 2.3. Correlation (rs) between the hormones (healthy foals only, n = 19) ................ 22
Table 2.4. Correlation (rs) between blood glucose and triglyceride concentrations,
QUICKI and hormones (all sick/hospitalized foals, n = 106).......................................... 22
Table 2.5. Correlation (rs) between the hormones (all sick/hospitalized foals, n = 106) .. 22
Table 2.6. Correlation (rs) between blood glucose and triglyceride concentrations,
QUICKI and hormones (sick non-septic foals, n = 62) ................................................... 23
Table 2.7. Correlation (rs) between the hormones (sick non-septic foals, n = 62) ............ 23
Table 2.8. Correlation (rs) between blood glucose and triglyceride concentrations,
QUICKI and hormones (septic foals only, n = 44) ......................................................... 24
Table 2.9. Correlation (rs) between the hormones (septic foals only, n = 44) .................. 24
Table 2.10. Comparison of serum glucose, triglyceride, hormone concentrations and
QUICKI between surviving and non-surviving septic neonatal foals at admission.......... 25
Table 2.11. Correlation (rs) between sepsis score and hormones/laboratory parameters .. 25
Table 2.12. Results for the final model of the multivariate logistic regression for risk
factors associated with non-survival in neonatal foals. ................................................... 26
ix
Table 3.1. Ghrelin, GH, serum glucose and triglyceride concentrations in neonatal foals at
admission. ..................................................................................................................... 37
Table 3.2. Correlation (rs) between ghrelin, GH, serum glucose and triglyceride
concentrations (healthy foals, n = 19) ............................................................................ 37
Table 3.3.Correlation (rs) between ghrelin, GH, serum glucose and triglyceride
concentrations (sick non-septic foals, n = 62)...................................................................2
Table 3.4. Correlation (rs) between ghrelin, GH, serum glucose and triglyceride
concentrations (septic foals, n = 44) .................................................................................2
Table 3.5. Comparison of ghrelin GH, serum glucose and triglycerides between surviving
and non-surviving septic neonatal foals at admission .......................................................2
Table 3.6. Correlation (rs) between sepsis score and ghrelin, GH, glucose and
triglycerides .....................................................................................................................3
x
List of Figures
Figure 2.1. Serum insulin and glucose concentrations in septic foals at admission.......... 27
xi
Chapter 1: Introduction and Literature Review
Sepsis is the most common cause of mortality in foals causing a significant impact on the
equine industry.1-4 Septic foals often present to intensive care units with anorexia and
hypoglycemia. Feed intake and energy metabolism are regulated by the endocrine
system, but endocrinopathies are a common sequela of sepsis and have become a major
focus of research in critically ill foals.5-8 We believe that disturbances of the energy
metabolism in septic neonatal foals are due to sepsis-mediated dysregulation of the
endocrine system, but limited information on the pathophysiology of hormonal regulation
in septic foals is available.
Maturation of the endocrine system and the associated energy metabolism in the equine
neonate is delayed and continues in the post-natal period.6;9-11 Neonates are highly
dependent on glucose intake and the endocrine glucoregulatory mechanisms are not
always fully competent at birth; thus, episodes of hypoglycemia are common in critically
ill foals.10-12 For example, newborn and premature foals have a lower insulin response to
increased blood glucose concentrations than older foals and foals born at term. 9;13 Postnatal maladaptation and illness are common in foals, making them prone to various
diseases, including sepsis. Sepsis is the most common cause of mortality in foals,
resulting in major economical losses to the equine industry. 1-4
Septic foals often present to intensive care units with anorexia and hypoglycemia, and
rapid intervention with energy-containing fluids is frequently indicated due to their
1
inability to nurse or intolerance to enteral feeding. A major complication of parenteral
nutrition in foals, including administration of dextrose with intravenous fluids, is the
development of hyperglycemia (e.g. glucose intolerance).14;15 Hyperglycemia has been
associated with increased mortality in critically ill humans16 due to oxidative stress,
glucotoxicity and β-cell dysfunction.17
Endocrinopathies have become a major focus of research in critically ill humans, and
more recently in foals.6-8 Insulin resistance and hyperglycemia are common
manifestations of endocrine dysregulation in people with sepsis or endotoxemia, 18-21 and
tight glycemic control utilizing insulin therapy has been shown to increase survival in
these patients.22 Similarly, insulin is frequently used in equine neonates when
hyperglycemia occurs,23 however, limited information on the hormonal control of energy
metabolism in septic neonatal foals is available.
Insulin is essential for energy regulation; it increases cellular glucose uptake,
glycogenesis, fatty acid synthesis, and potassium cellular uptake, decreases proteinolysis,
lipolysis and gluconeogenesis, and is a vasodilator to microcirculation. Insulin also has
anti-inflammatory properties by decreasing inflammatory cytokines and enhancing antiinflammatory mediators,19 which may confer some therapeutic benefit in patients in a
pro-inflammatory state (e.g. septicemia/endotoxemia). Insulin resistance is a common
feature of sepsis and endotoxemia in humans22;24 and has also been reported in horses.25;26
The quantitative insulin sensitivity check index, QUICKI, is a novel index of insulin
sensitivity based on a single fasting blood sample. 27 QUICKI provides a reproducible and
robust estimate of insulin sensitivity in humans, as it has strong correlations with insulin
2
sensitivity indices calculated from standard glucose clamp or FSIVGTT (frequently
sampled intravenous glucose tolerance test) studies in humans, and also appears useful in
rodents.28 QUICKI is a reliable index in insulin-resistant subjects.29 The authors did not
find any reference on the use of QUICKI in the equine species.
Glucagon is secreted by the α-cells of the pancreas and has opposing physiologic roles to
insulin as it stimulates gluconeogenesis, glycogenolysis and lipolysis.
Hyperglucagonemia has been reported in septic and endotoxemic humans as well as in
endotoxemic dogs and septic rats.30-32 Glucagon is thought to be important to maintain or
enhance gluconeogenesis in the catabolic state of critical illness.32
Leptin, an adipocyte-derived hormone (adipokine), is considered the main regulator of
satiety and its blood concentrations have been correlated with total body fat in horses,
humans, dogs and other species.33-36 Leptin concentrations decrease in feed restricted
mares,37 increase following a meal in humans38 and leptin increases insulin sensitivity in
humans.34 In addition to energy homeostasis, leptin has immunomodulatory properties,39
its synthesis is stimulated by inflammatory cytokines40 and leptin has been described as
an acute phase reactant.41;42 Increased leptin concentrations have been associated with
sepsis and endotoxemia in adult humans, rodents and dogs.42-47 However, an increase of
leptin concentrations was neither shown in septic human neonates, 48 nor in sheep,
cattle49;50 or pigs51 during experimental endotoxemia. No information is available on
leptin in horses (foals) with septicemia or endotoxemia.
Adiponectin, another adipokine, is negatively correlated with body fat mass in horses and
other species,33;34 yet also increases insulin sensitivity.52 Adiponectin promotes the cell
3
membrane translocation GLUT4, increases glycolysis and fatty acid oxidation. 53 Studies
in mice and humans have shown potential anti-inflammatory and LPS-neutralizing
properties of adiponectin.52;54;55
Ghrelin is produced by gastric ghrelin cells, which are oxyntic cells of the stomach that
are characterized by round, compact, electrondense secretory granules of the P/D 1 type in
humans, A-like type in the rat and X type in the dog, also referred to as X/A-like
cells.56;57 Ghrelin acts via the vagus nerve and is mainly orexigenic (increases food
intake). Upon food intake ghrelin release is suppressed by glucose, thus serum levels
drop after a meal.56 However, it is still unclear whether glucose directly or insulin causes
the inhibition of ghrelin secretion.58 On the other hand, ghrelin secretion increases with
anorexia. Another important function of ghrelin is that it stimulates the secretion of
growth hormone from the pituitary gland.
Growth hormone (GH) is a predominantly anabolic hormone that stimulates cell
reproduction and growth mediated by Insulin-like growth factors. GH additionally has
direct catabolic effects, e.g. enhanced lipolysis in fat cells and anti-insulin activity
causing restriction of glucose transport into cells.59
Ghrelin is one of the first hormones to rapidly increase in human and canine endotoxemia
and sepsis.60-62 Likewise, increased GH secretion has been observed in sepsis and
endotoxemia in humans and rodents.63-65 Ghrelin’s potential to down-regulate
proinflammatory cytokines and inhibit NF-κB has been studied in vitro 66 and in vivo
.67;68 The protective effect of ghrelin in sepsis and endotoxemia has become a major focus
of recent research in critically ill humans, especially in regards to treatment with
4
ghrelin.67;69;70 To the author’s knowledge, no information is available on ghrelin and
growth hormone in critically ill foals.
The purpose of this study was to investigate endocrine aspects of energy metabolism in
critically ill foals by determining serum/plasma concentrations of insulin, glucagon,
leptin and adiponectin, as well as ghrelin and growth hormone, in healthy, sick non-septic
and septic foals, as well as their association with serum glucose and triglyceride
concentrations, severity of disease (sepsis score) and outcome (death/survival). We
hypothesized that blood concentrations of insulin, glucagon, leptin, ghrelin and growth
hormone would be higher while adiponectin would be lower in septic foals compared to
healthy controls. We also expected an association of the magnitude of these differences
with degree of sepsis and non-survival.
5
Chapter 2: Insulin, glucagon, leptin and adiponectin in critically ill foals
2.1
Materials and Methods
Animals
Foals ≤7 days old of any breed or sex admitted to The Ohio State University Galbreath
Equine Center (OSU) and Hagyard Equine Medicine Institute (HEMI) during foaling
season of 2008 were included. Hospitalized foals were classified into one of the two
groups: sick non-septic and septic foals. Foals in the septic group had a sepsis score of
≥12 and/or a positive blood culture.71 Foals in the sick non-septic group were hospitalized
for illnesses other than sepsis (e.g. meconium impaction, hypoxic ischemic
encephalopathy, failure of transfer of passive immunity, flexural deformities) requiring
hospitalization. These foals had negative blood cultures and a sepsis score of ≤11. The
control group consisted of 18-24 hours old foals examined on a routine basis at breeding
farms in Kentucky. Foals included in the control group were born at the farm and were
clinically healthy based on physical exam, a normal complete blood count (CBC),
biochemistry profile, a serum immunoglobulin G (IgG) concentration >800 mg/dL and a
sepsis score of ≤4. Foals with a history of receiving glucose-containing fluids or
corticosteroids prior to admission to the hospital were excluded from the study.
6
Any foal that was discharged from the hospital was defined as a survivor. Foals that died
or were euthanized due to a grave medical prognosis were defined as non-survivors.
Individuals euthanized for other reasons such as financial constraints were excluded from
the study.
This study was approved by the Ohio State University Veterinary Teaching Hospital
executive committee, the Institutional Animal Care and Use Committee, and adheres to
the principles of humane treatment of animals in veterinary clinical investigations as
stated by the American College of Veterinary Internal Medicine and National Institute of
Health guidelines.
Clinical Information
History obtained upon admission included expected foaling date, duration of pregnancy,
parity, maternal illness, premature lactation, observed or assisted parturition, dystocia,
passing and appearance of the fetal membranes and medications (mare and foal). Clinical
data collected included signalment (sex, gestational and actual age, breed), physical
examination findings, CBC, biochemistry profile including serum glucose, fibrinogen, Llactate, IgG and triglyceride concentrations. For consistency, the sepsis score was
calculated by the first author for each foal individually, based on recorded history,
physical exam, and laboratory findings. 71
7
Sampling
Blood samples for hormone assays from foals admitted to both hospitals were obtained
on admission via sterile jugular venous catheterization. Blood was placed in plain serum
clot tubes and chilled aprotinin-EDTA tubes. Aprotinin was added to preserve sample
integrity by preventing potential protease degradation of hormones (500 kU/mL of whole
blood). The samples were stored in ice water and centrifuged within 12 hours at 5 °C,
2,000 g for 12 minutes. Serum and plasma were then aliquoted and stored at -80 °C until
analyzed. A 12 hour delay occurred in few samples, most samples were processed within
2 hours, and from human studies, there is no evidence that sample storage significantly
affects stability of insulin, leptin, or adiponectin. Human leptin is stable for 1 week at
room temperature and months at 4 °C.72 Human adiponectin is stable for 2 weeks at 2-8
°C and 1 day at room temperature, according to GenWay Biotech, San Diego, CA
92121(Human Adiponectin ELISA). The processing and storage in this study were
performed similarly to routine processing of human samples for immunoassays.
Blood samples for CBCi, serum biochemistry and IgGii were processed immediately by
the respective in-house laboratories (HDM and OSU). Samples from healthy control foals
were obtained during routine newborn foal examinations at the farm and processed the
same day.
i
Cell-Dyn 3500R analyzer, Abbott Laboratories, Abbott Park, IL
ii
Boehringer Mannheim/Hitachi 911 system, Boehringer Mannheim Corporation,
Indianapolis, IN
8
Hormone Concentrations
Blood concentrations of insulin (serum), glucagon (plasma), and adiponectin (serum)
were determined using human radioimmunoassaysiii,iv,v while leptin (serum) was
measured with a multispecies leptin radioimmunoassay. vi All assays have previously been
validated for the equine species.33;37;73;74
Quantitative Insulin Sensitivity Check Index (QUICKI)
Insulin sensitivity was assessed using the quantitative insulin-sensitivity check index
QUICKI, calculated by following formula:
QUICKI = 1/(log glucose [mg/dl] + log insulin [µIU/ml])27
iii
Coat-A-Count® human insulin radioimmunoassay, Siemens Healthcare Diagnostics Los
Angeles, CA
iv
Coat-A-Count® human glucagon radioimmunoassay, Siemens Healthcare Diagnostics,
Los Angeles, CA
v
Human adiponectin radioimmunoassay, Linco®, Millipore, St. Charles, MO
vi
Multispecies leptin radioimmunoassay, Linco®, Millipore, St. Charles, MO
9
Data Analysis
Shapiro-Wilk statistic was used to assess the data normality. Only glucose and
adiponectin concentrations were normally distributed. The remainder of the data was not
normally distributed. Therefore, median and interquartile ranges were calculated for
continuous variables. Nonparametric comparisons between the groups were computed
with the Kruskal-Wallis statistic and a Dunn’s post-test to compare each group
individually, using a statistical software programvii. The Mann-Whitney-U test was
applied to compare survivors with non-survivors. Significance was set at P < 0.05. The
Spearman rank order (rs) was used to define correlations between variables viii.
Continuous variables were categorized by cutoff values based on distribution within a
group, and analyzed using logistic regression (procgenmod ix) for binomial distribution.
Crude odds ratios and 95% confidence intervals were determined based on categories.
The dependent variable was survival/non-survival. All variables were screened and any
variables with a P value <0.25 were tested in a forward and backward stepwise
multivariate logistic regression to determine a final model. The Hosmer and Lemeshow
Goodness-of-Fit was determined using proc logistic. 75 Variables that resulted in a P value
<0.05 were retained in the model.
vii
Prism, version 4.0a, GraphPad Software Inc, San Diego, CA
viii
SigmaStat 3.5, Systat, Chicago, IL
ix
SAS version 9.1, SAS Institute Inc, Cary, NC
10
2.2
Results
Study Population
A total of 125 neonatal foals were included, of which 106/125 were hospitalized and
19/125 were healthy foals. Forty-four/106 (41%) were classified as septic, 62/106 (58%)
as sick non-septic. Of the 44 septic foals, 30 foals (68%) survived to discharge from the
hospital and 32 had a positive blood culture (73%). The median age of all hospitalized
foals at admission was 12 hours (range: sick non-septic 1-168 hours; septic 1-192 hours).
Healthy controls were all between 18 and 24 hours old.
All healthy controls were Thoroughbreds (n=19). Breeds representing the group of
hospitalized foals included Thoroughbred (n=71), Quarter Horse (n = 11), Standardbred
(9), Appaloosa (4), Warmblood (3), Friesian (2), American Paint Horse (2), Arabian (1),
Gypsy Vanner (1), Percheron (1) and 1 mixed breed. Of the hospitalized foals 50 were
fillies and 56 were colts, however, 15/19 healthy controls were fillies.
Serum Glucose and Triglycerides
Serum glucose concentrations were significantly lower in septic foals compared to sick
non-septic and healthy foals (p<0.001). Septic and sick non-septic foals had significantly
higher serum triglyceride concentrations than the healthy controls (p<0.001, table 1).
Glucose and triglyceride concentrations were inversely correlated in all sick/hospitalized
foals of the study population as well as in septic foals only (tables 3a and 5a), but not in
healthy foals.
11
Insulin, Glucagon, Leptin and Adiponectin Concentrations
Septic foals had significantly lower insulin and higher glucagon concentrations than the
healthy foals (p<0.001). Glucagon concentrations were higher and insulin concentrations
were lower in sick non-septic than in healthy foals (p<0.05 and p<0.001, respectively).
Glucagon concentrations were also higher in septic than in sick non-septic foals (p<0.01).
Insulin was not different between septic and sick non-septic foals. There was no
difference in leptin and adiponectin concentrations between groups (table 1).
Glucagon was positively correlated with leptin in hospitalized foals and in septic foals
only (tables 3b and 5b). Leptin was negatively correlated with adiponectin in hospitalized
foals and in sick non-septic foals only (tables 3b and 4b). There were no correlations
between individual hormones in healthy foals (table 2b).
Association of Hormone Concentrations with Glucose and Triglycerides
Insulin concentrations were positively correlated with glucose concentrations in all
groups. However, glucagon was inversely correlated with glucose only in the healthy
controls. When data of all hospitalized foals (sick non-septic and septic) was analyzed, all
hormones were correlated with glucose (table 3a), but when sick non-septic foals and
septic foals were analyzed as individual groups, there was no association between
glucagon, leptin or adiponectin and glucose (tables 4a and 5a). Glucagon was positively
correlated with triglycerides in all groups of sick/hospitalized foals (tables 3a, 4a and 5a),
but not in healthy foals. Further, there was no correlation between insulin, leptin or
adiponectin and triglycerides in any of the groups.
12
Association of Glucose and Triglyceride Concentrations with Survival
Of all septic foals, 68% survived (30/44). Glucose concentrations were significantly
lower in non-survivors than in septic foals that survived. However, we found no
difference in triglyceride concentrations between septic non-survivors and septic
survivors. Serum triglyceride concentration was one variable that was retained in the final
logistic regression model. In the entire foal study population (healthy, sick non-septic and
septic foals), multivariate logistic regression showed that overall non-survival was more
likely with serum triglyceride concentrations lower than 60 mg/dl (p<0.01).
The eight septic foals with the highest glucose concentrations (147-329 mg/dL) survived.
Only two of these eight individuals had a higher insulin response than normo- or
hypoglycemic septic foals (figure 1).
Association of Hormone Concentrations with Survival
Among septic foals, we found significantly higher insulin concentrations in non-survivors
than in survivors (p<0.001). Septic foals that had insulin concentrations higher than 4
µIU/ml were more likely to die than those who had low insulin on admission (OR 6.0;
95% CI, 1.2–36.4).
Glucagon and adiponectin were not different between the groups. Leptin concentrations
were lower in septic foals that died (p<0.05, table 5). Foals with leptin concentrations
lower than 1.1 ng/ml were more likely to die (OR 9.8; 95% CI, 1.4–199.5). Likewise,
when compared to the whole study population, the likelihood of non-survival was higher
in foals with low leptin concentrations (OR 3.98; 95% CI, 1.1-18.7).
13
Insulin sensitivity (QUICKI)
QUICKI was higher in sick non-septic and septic foals than healthy foals (table 1), but
was not different between septic survivors and septic non-survivors (table 6).
QUICKI was negatively correlated with leptin in all groups (tables 2a, 3a, 4a, 5a). In all
hospitalized foals (sick non-septic and septic), QUICKI was also negatively correlated
with insulin concentrations (tables 3a, 4a, 5a). Further, QUICKI was positively correlated
with glucagon, adiponectin and triglycerides in septic foals as well as all hospitalized
foals (tables 3a and 5a).
Sepsis Score
The sepsis score correlated significantly with insulin, glucagon, triglycerides and
QUICKI; however, no correlation was found between the sepsis score and the adipokines
(table 7). The sepsis score was the second variable that was retained in the final logistic
regression model. In this study population, non-surviving foals had the higher sepsis
scores (p<0.01).
Multivariate Analysis
The final model is attached in table 7. The model includes three variables: triglyceride
concentrations, cold extremities (yes/no) and sepsis score (<12/≥12). The Hosmer and
Lemeshow Goodness-of-Fit test indicates that the data fit the model well (P = 0.98).
14
2.3
Discussion
In the current study we documented that endocrine response of energy metabolism in
critically ill foals is characterized by hypoglycemia, hypertriglyceridemia, low insulin
and high glucagon concentrations. Mortality in septic foals was associated with low
leptin and high insulin concentrations.
Septic foals had significantly lower blood glucose concentrations than sick non-septic
and healthy foals, which was not unexpected, as hypoglycemia is a common finding in
critically ill foals.4;15;71 Among septic foals, non-survivors had lower glucose
concentrations than survivors, which is in agreement with a recent study in critically ill
foals.15 We found hyperglycemia in few critically ill foals of this study, perhaps caused
by increases in cortisol and catecholamine concentrations from stress of transportation or
illness. Hyperglycemia has also been associated with mortality and normoglycemia with
survival in critically ill foals,8;15 but septic foals in our study that were hyperglycemic
(glucose >130 mg/dL) survived. The effect of prolonged hyperglycemia was not
evaluated in this study.
Serum triglyceride concentrations were elevated in hospitalized foals, further supporting
a metabolic response to the increased energy needs. As carbohydrate stores in foals are
limited, the high energy demand depletes the carbohydrate stores, leading to mobilization
of fat depots. Ultimately, when the liver cannot maintain glucose production from fatty
acids, blood triglyceride concentrations increase. Similarly, hypertriglyceridemia is the
main feature of altered fat metabolism in critically ill humans. 24 In calves, glucose
concentrations decrease and triglyceride concentrations increase in response to
15
administration of endotoxin or TNFα. 76 High TNFα concentrations have been measured
in septic foals77 and could have contributed to the hypoglycemia and
hypertriglyceridemia found in the septic foals of this study.
We hypothesized that insulin would be higher in critically ill foals, based on what has
been observed in adult horses and calves with experimental endotoxemia. 26;76 Sepsis and
endotoxemia induce insulin resistance in humans, 18 horses25 and mice.78 However, in our
study, insulin was significantly lower in septic and sick non-septic foals as compared to
healthy controls, and insulin and glucose were positively correlated in all groups of the
study population. This, as well as the inverse relationship between glucose and glucagon
indicates an appropriate physiological response of insulin and glucagon to blood glucose
concentrations and contrasts the aforementioned studies.
To our knowledge, this is the first study to document the role of glucagon in the
endocrine response to sepsis in foals. Higher glucagon concentrations in the septic foals
could be interpreted as a physiological response, as glucagon is a catabolic hormone that
stimulates gluconeogenesis. Increased glucagon concentrations have also been
documented in septic rats and dogs,31;32 and endotoxemic humans.30
Adiponectin does not appear to be relevant in sick foals, as we did not find differences in
adiponectin concentrations between any of the groups. Adiponectin has been studied in
healthy horses,33;79;80 but no information is available in pathological conditions of adult
horses or foals. Adiponectin increases in human endotoxemia, 44;52 but decreases in rats
with sepsis.54 Studies in rodents and humans have shown potential anti-inflammatory and
LPS-neutralizing properties of adiponectin.52;54;55
16
Based on multivariate logistic regression analysis of all foals of the study population, low
serum triglyceride concentrations were associated with non-survival, which likely
represents a decompensation in fat metabolism in severely affected individuals, in which
fat stores may be depleted. The wide range of triglyceride concentrations in nonsurvivors could have also accounted for the lack of statistical difference in triglyceride
concentrations between survivors and non survivors.
In septic foals of our study insulin concentrations did not exceed those of the healthy
controls; however, higher insulin concentrations were associated with increased mortality
in the septic foals, suggesting that hyperinsulinemia was a response to systemic
inflammation. Insulin dysregulation has been documented in humans with multi-organ
dysfunction syndrome secondary to systemic inflammatory response syndrome who
developed insulin resistance, hypertriglyceridemia, and finally insulin deficiency from
pancreatic β-cell exhaustion.24
Insulin resistance as a response to systemic inflammation has not been evaluated in
critically ill foals by glucose clamp or FSIVGTT, and it was not feasible to do in this
study. As an alternative to determine insulin sensitivity based on a single sample, we
calculated QUICKI. The fact that QUICKI was higher in septic foals compared to the
other groups and positively associated with the sepsis score was unanticipated. We were
expecting evidence of insulin resistance in septic foals, because insulin resistance is a
common feature of sepsis and endotoxemia in humans22;24 and has been reported in
endotoxemic horses.25;26
17
Insulin treatment to maintain normoglycemia has been shown to decrease mortality in the
human ICU,20;21 because hyperglycemia is more detrimental than hypoglycemia.21;22;24
Intensive glucose control was associated with increased mortality as compared to
conventional glucose control (glucose target of 81-108 mg/dL versus <180 mg/dL).16 As
tight glucose control remains controversial in human medicine, in foals with
hyperglycemia this concept should be based on equine-specific studies rather than
extrapolated from human studies. Nevertheless, insulin treatment is frequent practice in
critically ill foals, especially when prolonged hyperglycemia develops from parenteral
nutrition.14;23 In addition, to its glucoregulatory effects, insulin appears to have beneficial
anti-inflammatory properties, decreasing pro-inflammatory and increasing antiinflammatory mediators in humans.19 In our study, foals were not treated with exogenous
insulin, but interestingly, septic foals that had higher endogenous insulin concentrations
on admission were more likely to die. This suggests that the protective anti-inflammatory
properties of insulin may not apply to endogenous insulin in foals.
Non-surviving septic foals had lower leptin concentrations and were more likely to die
than those with higher leptin concentrations. This finding is in agreement with a study by
Arnalich et al, in which high leptin concentrations in septic human patients were
associated with survival.43 In contrast, leptin concentrations were higher in septic and
endotoxemic rodents,36 dogs46;47 and adult humans,43 as well as in septic children that did
not survive.81 However, other studies found no association between leptin concentrations
and septicemia, shock, multiorgan failure or severity of disease in human neonates,
children, or endotoxemic sheep and cows.43-46;48-50;81 The lower blood glucose and leptin
18
concentrations in non-surviving septic foals display a physiological response of leptin to
increased caloric demand, as has been shown in feed restricted mares. 37 A similar
relationship between leptin and glucose has been demonstrated in horses that had an
increase in first insulin and then leptin after feeding, indicating that insulin drives an
increase in leptin.82 Conversely, we did not find a correlation between insulin and leptin
concentrations. The leptin response to systemic inflammation or sepsis appears to be
variable among species, age groups and possibly individuals. Thus it is difficult to make
strong conclusions on leptin and its prognostic value in septic foals with the current data,
and additional research using foals with various levels of sepsis or a controlled study will
be necessary to further address this question.
Although there is evidence that leptin increases insulin sensitivity34, leptin concentrations
were negatively correlated with QUICKI in all groups. We conclude that the role of
leptin in septic foals is too complex to make any conclusions based on these findings.
One limitation of this study was the sex bias in the control group (15/19 foals were
fillies). It is unclear, how the hormones of energy metabolism are influenced by sex in the
neonatal period. In sexual maturity, leptin has been shown to be influenced by sex
hormones83, however, in the neonatal period it is unlikely that sex hormones play a
significant role in hormonal regulation. Another limitation is the use of the sepsis score to
define sepsis in this study. Hypoglycemia is one of the variables used to calculate the
sepsis score and this has been an accepted approach in the current literature. In regards to
hypoglycemia, this may have caused a bias in the analysis. Of the foals defined as septic
in this study, 73% had a positive blood culture, so in only 27% of the foals classified as
19
septic by sepsis score only this approach may have confounded the results. Further,
QUICKI has not been assessed in horses before. To validate QUICKI in horses,
calculated QUICKI should be compared to results of FSIVGTT or hyperinsulinemic
euglycemic clamp methods in this species.
We documented numerous expected physiological changes in hormonal regulation of
energy metabolism in septic foals. But we also encountered unexpected concentrations of
leptin and insulin compared to how they control energy metabolism and hunger in health.
We do not believe that insulin has protective properties in foals as described for other
species, because higher insulin concentrations were associated with non-survival.
Controlled interventional studies are needed to determine the benefit of insulin treatment
in neonatal foals. This study shows that extrapolating evidence from the human literature
may not be appropriate with regards to insulin in critically ill equine patients. QUICKI
could be a valuable tool to assess insulin sensitivity in foals and horses, but additional
work would be required. Leptin seems to be involved in the systemic inflammatory
response in foal sepsis and could be a potential prognostic indicator of death in septic
foals when concentrations are low.
20
Table 2.1. Serum glucose, triglyceride, hormone concentrations and QUICKI in neonatal
foals at admission (n = 125)
Variable
Healthy
Sick non-septic
Septic
(n = 19)
(n = 62)
(n = 44)
145 (110-182)a
133 (34-272)a
87 (3-329)b
Triglycerides (mg/dl)
27 (19-56)a
40 (8-274)b
79 (12-986)b
Insulin (µIU/ml)
4.9 (3.2-13)a
2.7 (0.04-17)b
2.2 (0.04-16)b
Glucagon (pg/ml)
86 (24-279)a
170 (5.1-929)b
810 (8.6-1357)c
Leptin (ng/ml)
1.4 (0.55-5.6)
1.2 (0.14-6.3)
1.3 (0.20-1.9)
Adiponectin (ng/ml)
700 (517-852)
Glucose (mg/dl)
QUICKI
0.35 (0.30-0.38)
a
725 (443-1037)
792 (372-1008)
a
0.46 (0.23-6.3)b
0.39 (0.27-2.9)
Values expressed as median and range; different superscripts letter denote statistical differences, p<0.05
Table 2.2. Correlation (rs) between blood glucose and triglyceride concentrations,
QUICKI and hormones (healthy foals only, n = 19)
Variable
Glucose
Triglycerides
QUICKI
rs
P value
rs
P value
rs
P value
Insulin
0.41
0.039*
-0.32
0.186
0.16
0.507
Glucagon
-0.52
0.023*
0.12
0.631
-0.01
0.974
Leptin
-0.18
0.462
0.12
0.618
-0.81
0.001*
Adiponectin
0.03
0.894
0.23
0.352
-0.37
0.114
Triglycerides
-0.02
0.354
-
-
-0.08
0.748
*p<0.05; ¶Insulin was used to calculate QUICKI
21
Table 2.3. Correlation (rs) between the hormones (healthy foals only, n = 19)
Variable
Insulin
Glucagon
Leptin
rs
P value
rs
P value
rs
P value
Glucagon
-0.33
0.175
-
-
-
-
Leptin
-0.23
0.338
-0.33
0.175
-
-
Adiponectin
-0.31
0.193
0.26
0.286
0.32
0.182
*p<0.05
Table 2.4. Correlation (rs) between blood glucose and triglyceride concentrations,
QUICKI and hormones (all sick/hospitalized foals, n = 106)
Variable
Glucose
Triglycerides
QUICKI
rs
P value
rs
P value
rs
P value
Insulin
0.33
<0.001*
0.16
0.110
-0.25¶
0.012*¶
Glucagon
-0.20
0.045*
0.55
0.001*
0.36
0.002*
Leptin
0.24
0.01*
0.11
0.247
-0.32
0.001*
Adiponectin
-0.27
0.006*
0.15
0.141
0.29
0.001*
Triglycerides
-0.30
0.001*
-
-
0.23
0.023*
*p<0.05; ¶Insulin was used to calculate QUICKI
Table 2.5. Correlation (rs) between the hormones (all sick/hospitalized foals, n = 106)
Variable
Insulin
Glucagon
Leptin
rs
P value
rs
P value
rs
P value
Glucagon
0.08
0.430
-
-
-
-
Leptin
0.04
0.673
0.208
0.033*
-
-
Adiponectin
-0.01
0.937
-0.032
0.747
-0.427
0.001*
*p<0.05
22
Table 2.6. Correlation (rs) between blood glucose and triglyceride concentrations,
QUICKI and hormones (sick non-septic foals, n = 62)
Variable
Glucose
Triglycerides
QUICKI
rs
P value
rs
P value
rs
P value
Insulin
0.34
0.006*
-0.15
0.240
-0.30¶
0.017*¶
Glucagon
0.03
0.806
0.37
0.002*
-0.10
0.424
Leptin
0.23
0.067
0.17
0.166
-0.63
0.001*
Adiponectin
-0.21
0.095
-0.03
0.807
0.36
0.004*
Triglycerides
-0.09
0.483
-
-
-0.13
0.304
¶
*p<0.05; Insulin was used to calculate QUICKI
Table 2.7. Correlation (rs) between the hormones (sick non-septic foals, n = 62)
Variable
Insulin
Glucagon
Leptin
rs
P value
rs
P value
rs
P value
Glucagon
0.050
0.728
-
-
-
-
Leptin
0.14
0.265
0.21
0.096
-
-
Adiponectin
-0.24
0.062
-0.22
0.079
-0.41
0.001*
*p<0.05
23
Table 2.8. Correlation (rs) between blood glucose and triglyceride concentrations,
QUICKI and hormones (septic foals only, n = 44)
Variable
Glucose
Triglycerides
QUICKI
rs
P value
rs
P value
rs
P value
Insulin
0.56
<0.001*
0.23
0.145
-0.15¶
0.342¶
Glucagon
-0.04
0.798
0.56
<0.001*
0.38
0.015*
Leptin
0.20
0.190
0.02
0.869
-0.56
0.001*
Adiponectin
-0.29
0.064
0.21
0.180
0.33
0.040*
Triglycerides
-0.40
0.008*
-
-
0.58
0.001*
¶
*p<0.05; Insulin was used to calculate QUICKI
Table 2.9. Correlation (rs) between the hormones (septic foals only, n = 44)
Variable
Insulin
Glucagon
Leptin
rs
P value
rs
P value
rs
P value
Glucagon
0.25
0.101
-
-
-
-
Leptin
-0.12
0.462
0.35
0.021*
-
-
Adiponectin
-0.13
0.397
0.25
0.101
-0.26
0.097
*p<0.05
24
Table 2.10. Comparison of serum glucose, triglyceride, hormone concentrations and
QUICKI between surviving and non-surviving septic neonatal foals at admission
Variable
Survivors
Non-survivors
(n = 30)
(n = 14)
Glucose (mg/dl)
109 (24-329)
40 (3.0-138)
0.013*
Triglycerides (mg/dl)
159 (12-986)
75 (15-588)
0.780
Insulin (µIU/ml)
1.6 (0.04-13.3)
3.9 (0.04-15.6)
0.048*
Glucagon (pg/ml)
566 (23-1228)
516 (8.6-1357)
0.749
1.3 (0.3-1.8)
1.0 (0.2-1.7)
0.023*
Adiponectin (ng/ml)
770 (372-1008)
800 (660-985)
0.457
QUICKI
0.44 (0.23-6.3)
0.50 (0.36-2.9)
0.597
Leptin (ng/ml)
P value
Values expressed as median and range, *p<0.05
Table 2.11. Correlation (rs) between sepsis score and hormones/laboratory parameters
Sepsis Score
rs
P value
Insulin
-0.18
0.031*
Glucagon
0.35
<0.0001*
Leptin
-0.15
0.123
Adiponectin
0.17
0.077
Glucose
-0.47
<0.0001*¶
Triglycerides
0.46
0.0002*
QUICKI
0.04
0.001*
¶
*p<0.05; Glucose was used to calculate sepsis score
25
Table 2.12. Results for the final model of the multivariate logistic regression for risk
factors associated with non-survival in neonatal foals.
Variable
OR
95% CI
Triglycerides (mg/dl)
P value
<0.05
<30
98.4
7.0-3585.3
31- 60
45.8
2.0-2205.2
>61
Reference
N/A
Cold extremities
<0.01
no
0.02
0.02-0.22
yes
Reference
N/A
Sepsis score
<0.01
<12
0.04
0.03-0.54
≥12
Reference
N/A
OR = Odds ratio, 95% CI = 95% Confidence interval, Reference = Reference group for comparison, N/A = Not
applicable
26
100
50
Insulin (uIU/mL)
20
15
10
5
0
0
50
100
150
200
250 300 400
Glucose (mg/dL)
Figure 2.1. Serum insulin and glucose concentrations in 30 septic surviving () and 14
septic non-surviving () neonatal foals at admission. The eight septic foals with the
highest glucose concentrations (147-329 mg/dL, on right to the dashed line) were
survivors.
27
Chapter 3: Ghrelin and Growth Hormone in Critically Ill Foals
3.1
Material and Methods
Animals
Foals ≤7 days old of any breed or sex admitted to The Ohio State University Galbreath
Equine Center (OSU) and Hagyard Equine Medicine Institute (HEMI) during foaling
season of 2008 were included. Hospitalized foals were classified into one of the two
groups: sick non-septic and septic foals. Foals in the septic group had a sepsis score of
≥12 and/or a positive blood culture.71 Foals in the sick non-septic group were hospitalized
for illnesses other than sepsis (e.g. meconium impaction, hypoxic ischemic
encephalopathy, failure of transfer of passive immunity, flexural deformities) requiring
hospitalization. These foals had negative blood cultures and a sepsis score of ≤11. The
control group consisted of 18-24 hours old foals examined on a routine basis at breeding
farms in Kentucky. Foals included in the control group were born at the farm and were
clinically healthy based on physical exam, a normal complete blood count (CBC),
biochemistry profile, a serum immunoglobulin G (IgG) concentration >800 mg/dL and a
sepsis score of ≤4. Foals with a history of receiving glucose-containing fluids or
corticosteroids prior to admission to the hospital were excluded from the study.
Any foal that was discharged from the hospital was defined as a survivor. Foals that died
or were euthanized due to a grave medical prognosis were defined as non-survivors.
28
Individuals euthanized for other reasons such as financial constraints were excluded from
the study.
This study was approved by the Ohio State University Veterinary Teaching Hospital
executive committee, the Institutional Animal Care and Use Committee, and adheres to
the principles of humane treatment of animals in veterinary clinical investigations as
stated by the American College of Veterinary Internal Medicine and National Institute of
Health guidelines.
Clinical Information
History obtained upon admission included expected foaling date, duration of pregnancy,
parity, maternal illness, premature lactation, observed or assisted parturition, dystocia,
passing and appearance of the fetal membranes and medications (mare and foal). Clinical
data collected included signalment (sex, gestational and actual age, breed), physical
examination findings, CBC, biochemistry profile including serum glucose, fibrinogen, Llactate, IgG and triglyceride concentrations. For consistency, the sepsis score was
calculated by the first author for each foal individually, based on recorded history,
physical exam, and laboratory findings. 71
Sampling
Blood samples for hormone assays from foals admitted to both hospitals were obtained
on admission via sterile jugular venous catheterization. Blood was placed in plain serum
clot tubes and chilled aprotinin-EDTA tubes. Aprotinin was added to preserve sample
29
integrity by preventing potential protease degradation of hormones (500 kU/mL of whole
blood). The samples were stored in ice water and centrifuged within few hours at 5 °C,
2,000 g for 12 minutes. Serum and plasma were then aliquoted and stored at -80 °C until
analyzed. Blood samples for CBC x, serum biochemistry and IgGxi were processed
immediately. Samples from healthy control foals were obtained during routine newborn
foal examinations at the farm and processed the same day.
Hormone Concentrations
Plasma ghrelin and GH concentrations were determined using an active ghrelin
radioimmunoassayxii and a porcine/canine growth hormone radioimmunoassayxiii. Both
assays have previously been validated for the equine species.84;85
Data Analysis
Shapiro-Wilk statistic was used to assess the data normality. The data was not normally
distributed. Therefore, median and interquartile ranges were calculated for continuous
variables. Nonparametric comparisons between the groups were computed with the
Kruskal-Wallis statistic and a Dunn’s post-test to compare each group individually, using
x
Cell-Dyn 3500R analyzer, Abbott Laboratories, Abbott Park, IL
xi
Boehringer Mannheim/Hitachi 911 system, Boehringer Mannheim Corporation,
Indianapolis, IN
xii
Ghrelin (active) radioimmunoassay, Linco®, Millipore, St. Charles, MO
xiii
Porcine/canine growth hormone radioimmunoassay, Linco®, Millipore, St. Charles,
MO
30
a statistical software programxiv. The Mann-Whitney-U test was applied to compare
survivors with non-survivors. Significance was set at P < 0.05. The Spearman rank order
(rs) was used to define correlations between variables xv. Continuous variables were
categorized by cutoff values based on distribution within a group, and analyzed using
logistic regression (procgenmodxvi) for binomial distribution. Crude odds ratios and 95%
confidence intervals were determined based on categories. The dependent variable was
survival/non-survival. All variables were screened and any variables with a P value <0.25
were tested in a forward and backward stepwise multivariate logistic regression to
determine a final model. The Hosmer and Lemeshow Goodness-of-Fit was determined
using proc logistic.75 Variables that resulted in a P value <0.05 were retained in the
model.
3.2
Results
Study Population
A total of 125 neonatal foals were included, of which 106/125 were hospitalized and
19/125 were healthy foals. Forty-four/106 (41%) were classified as septic, 62/106 (58%)
as sick non-septic. Of the 44 septic foals, 30 foals (68%) survived to discharge from the
hospital and 32 had a positive blood culture (73%). The median age of all hospitalized
xiv
xv
Prism, version 4.0a, GraphPad Software Inc, San Diego, CA
SigmaStat 3.5, Systat, Chicago, IL
xvi
SAS version 9.1, SAS Institute Inc, Cary, NC
31
foals at admission was 12 hours (range: sick non-septic 1-168 hours; septic 1-192 hours).
Healthy controls were all between 18 and 24 hours old.
All healthy controls were Thoroughbreds (n=19). Breeds representing the group of
hospitalized foals included Thoroughbred (n=71), Quarter Horse (n = 11), Standardbred
(9), Appaloosa (4), Warmblood (3), Friesian (2), American Paint Horse (2), Arabian (1),
Gypsy Vanner (1), Percheron (1) and 1 mixed breed. Of the hospitalized foals 50 were
fillies and 56 were colts, however, 15/19 healthy controls were fillies.
Serum Glucose and Triglycerides
Serum glucose concentrations were significantly lower in septic foals compared to sick
non-septic and healthy foals (p<0.001). Septic and sick non-septic foals had significantly
higher serum triglyceride concentrations than the healthy controls (p<0.001, table 1).
Glucose and triglyceride concentrations were inversely correlated in septic foals (table 4),
but not in healthy foals (table 2).
Association of Glucose and Triglyceride Concentrations with Survival
Of all septic foals, 68% survived (30/44). Glucose concentrations were significantly
lower in non-survivors than in septic foals that survived. However, we found no
difference in triglyceride concentrations between septic non-survivors and septic
survivors. Serum triglyceride concentration was one variable that was retained in the final
logistic regression model. In the entire foal study population (healthy, sick non-septic and
32
septic foals), multivariate logistic regression showed that overall non-survival was more
likely with serum triglyceride concentrations lower than 60 mg/dl (p<0.01).
Ghrelin and Growth Hormone Concentrations
Septic foals had significantly higher ghrelin concentrations than sick non-septic foals and
healthy controls (p<0.001). GH concentrations were higher in septic foals and sick nonseptic foals compared to healthy foals (p<0.05, table 1).
Association of Hormone Concentrations with Glucose and Triglycerides
Ghrelin and GH were negatively correlated with glucose in healthy foals (table 2). In
septic foals, ghrelin was positively correlated with triglycerides, and GH was negatively
correlated with glucose (table 4). Ghrelin and GH were not correlated in any group.
Association of Hormone Concentrations with Survival
There was no statistical difference in ghrelin or growth hormone concentrations between
septic survivors and non-survivors (table 5). However, logistic regression showed that
foals with ghrelin concentrations lower than 15 pg/ml were less likely to die (OR 0.23;
95% CI, 0.05-0.79) than foals with concentrations higher than 15 pg/ml. Likewise, the
likelihood of non-survival was lower in foals with GH concentrations less than 1.8ng/ml
(OR 0.21; 95% CI, 0.05-0.73).
33
Sepsis Score
The sepsis score correlated significantly with ghrelin and triglyceride concentrations
(positive correlation), as well as glucose (negative correlation). No correlation was found
between the sepsis score and GH (table 6). The sepsis score was the second variable that
was retained in the final logistic regression model. In this study population, non-surviving
foals had the higher sepsis scores (p<0.01).
Multivariate Analysis
The final model is described in table 7. The model includes three variables: triglyceride
concentrations, cold extremities (yes/no) and sepsis score (<12/≥12). The Hosmer and
Lemeshow Goodness-of-Fit test indicates that the data fit the model well (P = 0.98).
3.3
Discussion
In the current study we documented that the endocrine response of energy metabolism in
critically ill foals is characterized by hypoglycemia, hypertriglyceridemia, and an
increase in ghrelin and GH concentrations. Increases in ghrelin and GH were associated
with decreased serum glucose concentrations and increased serum triglyceride
concentrations.
Ghrelin secretion physiologically increases when blood glucose concentrations are low,
so the observed association between glucose and ghrelin appears like an appropriate
physiological response. The higher ghrelin concentrations in septic foals and the
correlation with the sepsis score are in agreement with findings in human abdominal
34
sepsis60 and endotoxemia.61 Ghrelin levels also increased in dogs after administration of
endotoxin,62 but studies in rats remain contradictory,86-89 Negative energy balance and
anorexia provide an explanation for the increase in plasma ghrelin observed in septic
foals. Reduced ghrelin clearance due to hepatorenal injury in sepsis and endotoxemia62;90
could be another reason for the observed increase of ghrelin. Ghrelin has recently been
called “a signal of insufficient energy intake”91 and obviously plays a significant role in
negative energy balance. In critically ill foals negative energy balance develops from
anorexia in conjunction with the higher energy demand of illness, often reflected as
hypoglycemia. In humans, ghrelin induces lipolysis in adipocytes.92 Assuming that the
increase in plasma ghrelin, likely stimulated by hypoglycemia similarly promotes
lipolysis in horses, ghrelin may be pivotal in the observed increase in serum triglycerides
in septic foals.
In mice, blood triglycerides promoted the transport of intravenously administered ghrelin
across the blood-brain-barrier, and fasting also tended to promote ghrelin transport across
the blood-brain-barrier.93 Possibly, endogenous ghrelin has a similar effect as exogenous
ghrelin, and not only hypoglycemia results in increased ghrelin secretion, but
subsequently hypertriglyceridemia contributes to the central signaling of ghrelin to
induce hunger and stimulate feed intake. Nevertheless, in critically ill neonatal foals,
these proposed mechanisms apparently are not able to overcome anorexia, and other
mechanisms and mediators of anorexia still predominate.
Ghrelin is not only a GH secretagogue and orexigenic factor, ghrelin additionally has
significant anti-inflammatory properties and is being studied as a treatment for sepsis and
35
endotoxemia in rodent models. Ghrelin down-regulates proinflammatory cytokines and
inhibits NF-κB in vitro66 and in vivo.67;68 To the author’s knowledge, the antiinflammatory properties of ghrelin have not been studied in septic or endotoxemic
domestic mammals other than rodents. Further research is needed to determine the
possible beneficial effects of exogenous ghrelin in critically ill foals.
The increased GH concentration in septic foals was possibly due to the effect of increased
ghrelin, although there was no significant correlation between ghrelin and GH. This
finding was unexpected, because ghrelin stimulates GH secretion. This leads to the
assumption that GH secretion is regulated by other mediators aside from ghrelin. The
association between GH secretion and sepsis or endotoxemia has been studied in several
species. Recombinant bovine TNF has been shown to mediate GH secretion in dairy
heifers,94 and GH secretion is stimulated in human sepsis due to an abnormal pituitary
response.64
Foals with low GH concentrations were overall less likely to die, but the lack of statistical
difference between GH concentrations of septic survivors and non-survivors questions
the usefulness of GH as a predictor for mortality in critically ill foals. This is in contrast
to studies in septic children65 and adult critically ill patients.63
Hypoglycemia and hypertriglyceridemia are common in septic foals. Increases in ghrelin
and GH appear to be linked to the energy status in these foals. The observed increases in
ghrelin and GH in critically ill foals is in agreement with results of studies in other
species95 and humans, however, ghrelin and GH are not useful for prediction of mortality
36
in septic foals. Possible implications for treatment of septic foals with ghrelin should be
further investigated.
Table 3.1. Ghrelin, GH, serum glucose and triglyceride concentrations in neonatal foals at
admission.
Variable
Healthy
Sick Non-Septic
Septic
(n = 62)
(n = 44)
(n = 19)
Ghrelin (pg/ml)
7.8 (0.15-21)
a
8.9 (0.15-190)
GH (ng/ml)
1.6 (0.05-4.2)a
2.8 (0.05-35)b
4.9 (0.05-93)b
Glucose (mg/dl)
145 (110-182)a
133 (34-272)a
87 (3-329)b
27 (19-56)a
40 (8-274)b
79 (12-986)b
Triglycerides (mg/dl)
a
20 (0.15-779)b
Values expressed as median and range, different letter superscripts denote statistical difference, p<0.05
Table 3.2. Correlation (rs) between ghrelin, GH, serum glucose and triglyceride
concentrations (healthy foals, n = 19)
Variable
Glucose
Triglycerides
Growth Hormone
rs
P value
rs
P value
rs
P value
Ghrelin
-0.51
0.025*
0.11
0.654
0.013
0.957
GH
-0.49
0.034*
-0.03
0.906
-
-
Triglycerides
-0.02
0.354
-
-
-
*p<0.05
37
Table 3.3. Correlation (rs) between ghrelin, GH, serum glucose and triglyceride
concentrations (sick non-septic foals, n = 62)
Variable
Glucose
Triglycerides
Growth Hormone
rs
P value
rs
P value
rs
P value
Ghrelin
-0.13
0.314
0.004
0.975
0.04
0.788
GH
0.10
0.437
0.15
0.256
-
-
Triglycerides
-0.09
0.483
-
-
-
*p<0.05
Table 3.4. Correlation (rs) between ghrelin, GH, serum glucose and triglyceride
concentrations (septic foals, n = 44)
Variable
Glucose
Triglycerides
Growth Hormone
rs
P value
rs
P value
rs
P value
Ghrelin
-0.14
0.381
0.30
0.045*
0.18
0.243
GH
-0.39
0.009*
0.20
0.208
-
-
Triglycerides
-0.40
0.008*
-
-
-
*p<0.05
Table 3.5. Comparison of ghrelin GH, serum glucose and triglycerides between surviving
and non-surviving septic neonatal foals at admission
Variable
Survivors
Non-survivors
(n = 30)
(n = 14)
Ghrelin (pg/ml)
19.2 (0.15-779.2)
23.5 (2.13-476.1)
0.59
GH (ng/ml)
4.39 (0.31-93.0)
9.40 (0.05-91.0)
0.10
Glucose (mg/dl)
109 (24-329)
40 (3.0-138)
0.013*
Triglycerides (mg/dl)
159 (12-986)
75 (15-588)
0.78
Values expressed as median and range, *p<0.05
2
P value
Table 3.6. Correlation (rs) between sepsis score and ghrelin, GH, glucose and
triglycerides
Ghrelin
GH
Glucose
Triglycerides
Sepsis Score
rs
P value
0.42
<0.0001*
0.10
0.337
-0.47
<0.0001*¶
0.46
0.0002*
*p<0.05; ¶Glucose was used to calculate sepsis score
3
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